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首页> 外文期刊>Biochemistry >Sensitive monitoring of the dynamics of a membrane-bound transport protein by tryptophan phosphorescence spectroscopy.
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Sensitive monitoring of the dynamics of a membrane-bound transport protein by tryptophan phosphorescence spectroscopy.

机译：色氨酸磷光谱光谱法的膜结合蛋白质动力学的敏感性监测。

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This paper presents a tryptophan phosphorescence spectroscopy study on the membrane-bound mannitol transporter, EII(mtl), from E. coli. The protein contains four tryptophans at positions 30, 42, 109, and 117. Phosphorescence decays in buffer at 1 degrees C revealed large variations of the triplet lifetimes of the wild-type protein and four single-tryptophan-containing mutants. They ranged from <70 microseconds for the tryptophan at position 109 to 55 ms for the residue at position 30, attesting to widely different flexibilities of the tryptophan microenvironments. The decay of all tryptophans is multiexponential, reflecting multiple stable conformations of the protein. Both mannitol binding and enzyme phosphorylation had large effects on the triplet lifetimes. Mannitol binding induces a more ordered structure near the mannitol binding site, and the decay becomes significantly more homogeneous. In contrast, enzyme phosphorylation induces a large relaxation of the protein structure at the reporter sites. The implications of these structural changes on the coupling mechanism between the transport and the phosphorylation activity of EII(mtl) are discussed. Taken as a whole, our data show that tryptophan phosphorescence spectroscopy is a very sensitive technique to explore conformational dynamics in membrane proteins.
机译：本文介绍了大肠杆菌的膜结合甘露醇转运蛋白，EII（MTL）的色氨酸磷光光谱研究。该蛋白质在第30,42,109和117个处含有四种色氨酸。在1℃下缓冲液中的磷光衰减显示出野生型蛋白质的三重态寿命和含四种单色氨酸突变体的大的大变化。它们在109至55ms的位置30至55ms的70微秒内范围为位于第30位，证明了色氨酸微环境的广泛不同的灵活性。所有色氨酸的衰减是多因素，反映蛋白质的多重稳定构象。甘露醇结合和酶磷酸化两者都对三联寿命有很大影响。甘露醇结合在甘露醇结合位点附近诱导更有序的结构，衰减变得明显更加均匀。相比之下，酶磷酸化在报告部位诱导蛋白质结构的大量放松。讨论了这些结构变化对eII（MT1）的运输和磷酸化活性之间的偶联机制的影响。我们的数据表明，色氨酸磷光光谱是一种非常敏感的技术，用于探索膜蛋白中的构象动态。

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